Profile of Serum Heat Shock Protein-27 Level in Patients with Salivary Gland Tumor

Statement of the Problem: Heat shock protein 27 (HSP27) plays important roles in many cellular processes and has been implicated in different types of diseases such as cancers. Purpose: This study aimed to evaluate the serum level of HSP27 in patients with salivary gland tumors and to determine its possible correlation with the prognosis of the disease. Materials and Method: This cross-sectional study was performed on 60 patients with sali-vary gland tumor including 16 pleomorphic adenoma, 33 adenoid cystic carcinoma, 6 mu-coepidermoid carcinoma, 5 acinic cell carcinoma, and 28 healthy control subjects. The con-trol cases were healthy blood donors who matched the study group in age and sex. Serum samples were obtained from the clotted blood and HSP27concentrations were measured with sandwich enzyme-linked immunosorbent assay (ELISA). Statistical analysis was performed by using one-way ANOVA, post Hoc test, independent sample t-test, and ROC analysis. A p value of less than 0.05 was considered as significant. Results: The mean serum level of HSP27 was 3956.1±3830.1 (pg/ml) in patients with malig-nant salivary gland tumor, which was significantly higher than that in benign salivary gland tumor (752.2±485.6) and healthy controls (602.3±575.8) (p <0.001). However, there was no significant difference in the HSP27 serum levels between the patients with benign salivary gland tumors and healthy controls (p= 0.2). No association was detected between the mean serum levels of HSP27 and clinicopathologic factors such as age, sex, stage and nodal metas-tasis (p > 0.05), except for the tumor size (p= 0.04). Conclusion: The HSP27 concentration increased in patients with malignant salivary gland tumors. Moreover, the HSP27 level was correlated with tumor growth, invasiveness, and diagnosability. Yet, larger clinical studies are required to explore its prognostic value.


Introduction
Salivary gland tumors comprise 1-4% of human neoplasms [1]. Pleomorphic adenoma (PA), the most common benign salivary gland tumor, has an incidence of 70% [2]. Mucoepidermoid carcinoma (MEC) and adenoid cystic carcinoma (ACC) [3] are the most common sali-vary gland cancers that can metastasize to the regional lymphatics or distant organs. It has been shown that salivary gland tumors have numerous complexities in the site of occurrence, structural origins, histopathologic features, clinical appearance and prognosis [4][5]. Therefore, their mechanism of development and prognosis is significantly different [6]. Moreover, the salivary gland tumors have recurrence potential after treatment; thus, finding methods of early diagnosis and prevention of these tumors is very important in clinical cancers [6].
Heat shock proteins (HSPs) are a kind of important molecular chaperones expressed in the nuclei, cytoplasm, and cell membrane of both eukaryotic and prokaryotic organisms, which induce cell growth regulation and proliferation [7][8]. HSPs family is subdivided into four major families according to their activity including HSP 90, HSP 70 , HSP 60 , and small molecular weight HSPs such as HSP 27 with 27 KDa weight [9]. HSP 27 plays important roles in many cellular processes such as cell differentiation [8], inhibition of apoptosis [10], thermotolerance, cytoskeleton dynamics, and signal transduction [11]. HSP 27 has also been implicated in different types of diseases including cancers [12], renal injury and fibrosis [13], as well as cardiovascular and neurodegenerative diseases [14]. Over-expression of HSP 27 has been found in different tumor types and is involved in tumor formation and development, prognosis, treatment and biological behaviors [15][16]. Therefore, it can be a therapeutic target and potential biomarker for diagnosis and prognosis [12]. In a recent study by Vahen-Kepenekian et al. [17], it was demonstrated that HSPs overexpression in the cytosol of malignant transformed cells resulted in their translocation into the extracellular serum. The overexpression of HSP 27 in the serum might also be the result of its release following the stressinduced death of the necrotic cells [17].
Guilan et al. [6] showed the overexpression of HSP 27 in the cytoplasm of the tumoral epithelium in the salivary gland tissue and found a negative correlation between HSP 27 tissue level and the tumor size, invasion, and metastasis. Accordingly, this study was conducted to evaluate the serum level of HSP 27 in patients with salivary gland tumors and to determine if it has a correlation with prognosis or not. It seems that HSP 27 can be a novel target for increasing the effect of cancer treatment; therefore, this study was designed to assess and compare the serum level of HSP 27 in the most common benign and malignant salivary gland tumors and to consider whether its serum level was correlated with any tumor characteristics such as tumor grade and stage. Statistical analysis was performed by using one-way ANOVA, post hoc test, independent sample t-test, and ROC analysis. A p value of less than 0.05 was considered as significant. This yielded a sensitivity of 75% and specificity of 75%.  was significantly higher in patients with larger tumor size than the small tumor size.

Discussion
Biomarkers are used to detect and monitor the growth of the tumor and evaluate the effect of anti-cancer therapies [18]. A major disadvantage of tissue-based biomarkers is their limited accessibility and the risk of developing infection due to invasive surgery. Therefore, bloodderived biomarkers are better than biopsies in detecting and monitoring the tumor in that these samples can be taken before, during and after treatment with minimal invasion [19].
HSPs are a series of highly conserved proteins that play a crucial role in maintaining protein homeostasis [20]. HSPs are produced under stressful conditions and they participate in folding and assembling the proteins [12]. However, HSPs can be secreted in the blood circulation and have been shown to interact with some immune cells [21]. It has been demonstrated that HSP 27 expression is crucial for tumor development and is involved in tumor behavior and prognosis [22][23]. Increased HSP 27 expression has been reported in different cancers [24][25] and is associated with poor prognosis in the liver, prostate, and gastric carcinoma [26][27].
In the present study, circulation of HSP 27 level was obviously upregulated in patients with malignant Salivary gland tumors than in those with benign tumors and healthy control subjects. This was in line with those of the previous studies that evaluated the HSP 27 level in breast cancer, non-small cell lung cancer, pancreatic and gastric adenocarcinoma [28][29][30][31]. This study demonstrated that HSP 27 level can be use-d as a discriminating biomarker for differentiating patients with and without cancer. This was consistent with the results of Lia et al. [27] and De et al. [32][33]. Lia et al. [27] stated that Heat shock protein 27 levels were significantly higher in cancer and pancreatitis compared with control (p< 0.001 for both) and De et al. [32][33] reported that the use of HSP 27 ELISA could be extremely useful in evaluating the role of soluble HSP 27 in breast or other cancers.
Cell damage or necrotic cell death can cause the release of intracellular HSP into circulation that could lead to elevated HSP serum concentration [34]. In this study, HSP elevation in patients with malignant salivary gland tumor might be related to its increased production and release because of cell damage. In salivary gland carcinoma, HSP 27 level did not correlate with prognostic factors, except for the tumor size. In a recent study on non-small cell lung cancer, it was found that the serum level of HSP 27 was significantly elevated in patients at the advanced stage than the early stages [35]. However, no significant relationship was found between the serum level of HSP 27 and histological types and sex [35].
Van Eden et al. [36] showed that HSP released by the cells were biologically active molecule, confirming the hypothesis that elevated circulating HSP 27 [11] demonstrated that HSP 27 overexpression was associated with resistance to chemotherapy. Resistance to chemotherapy and radiotherapy has been reported in malignant salivary gland tumor [39]. Resistance to chemotherapy, in salivary gland cancer may partly contribute to elevated HSP 27 .
This study could not evaluate the expression of HSP 27 and the relationship between HSP 27 expression and its serum level. Therefore, further studies are required to explore this relationship.

Conclusion
This study showed that HSP 27 concentration increased in patients with malignant salivary gland tumors and besides its diagnostic ability, its level was correlated with tumor growth and invasiveness. Nevertheless, larger clinical studies are required to verify its prognostic value.